Project Summary/Abstract Influenza A virus (IAV) evolves rapidly in response to vaccines and drugs, causing significant public health and economic burdens. Interactions between mutations, or epistasis, determine how IAV will evolve. Different types of epistasis, including positive and negative, have different evolutionary consequences. However, little is known about the types of epistasis in IAV, limiting our ability to predict and control the virus. The long-term goal is to elucidate how basic evolutionary processes affect public health. The objectives of this project are to characterize pairwise epistasis between all 8 genomic segments of IAV and within the HA gene encoding the antigenic hemagglutinin protein. A tractable genetic system will allow the creation of thousands of double mutants using an existing genome-wide library of influenza viruses with defined point mutations. Using a novel, sensitive, and high-throughput fitness assay for viral growth, the fitness of each single and double mutant will be determined, and the magnitude and sign of epistasis will be quantified. Preliminary data indicate that this fitness assay has very low measurement error, enabling precise measurement of epistasis in thousands of mutants. The first aim is to characterize pairwise epistasis between all pairs of IAV segments. Based on theoretical predictions related to viral evolutionary constraints, the hypothesis is that epistasis in IAV is negative on average. Distinct epistatic patterns between segments may reveal previously unknown functional interactions between IAV segments. To complete this aim, the fitness of thousands of double and single mutants in all pairs of segments will be compared. The second aim is to characterize pairwise epistasis within HA. Based on previous work on protein- folding constraints, the hypothesis is that epistasis within HA is negative on average. To test this hypothesis, the fitness of hundreds of double and single mutants within HA will be compared. This project is innovative because, to our knowledge, it will be the only study of epistasis between random point mutations in different genes of IAV and almost a hundred times larger than the largest such study in a virus. This innovation is enabled by creative use of the IAV genetic engineering system and a novel fitness assay. This project is significant because characterizing the types of epistasis in IAV will improve predictions of the evolution of seasonal and pandemic strains and aid in the design of effective vaccines. Through completion of the proposed research project, mentorship, and other activities, the applicant will achieve his training goals in the fields of molecular evolution and population genetics, high-throughput methods and data analysis, effective scientific communication, and clinical medicine. This will enable the applicant to achieve his ultimate goal of becoming a physician-scientist who answers fundamental questions in evolutionary biology with practical implications for human health.